Circular RNA circ_DROSHA alleviates the neural damage in a cell model of temporal lobe epilepsy through regulating miR-106b-5p/MEF2C axis

Temporal lobe epilepsy (TLE) is the most prevalent form of acquired epilepsy. Circular RNAs (circRNAs) have recently been highlighted as important regulators in TLE. Nevertheless, the role and mechanism of circRNA Drosha ribonuclease III (circ_DROSHA) in TLE pathogenesis are still unknown. Magnesium-free extracellular solution was used to establish the TLE cell model. The levels of circ_DROSHA, myocyte-specific enhancer factor 2C (MEF2C) and miR-106b-5p were determined by qRT-PCR and western blot. Cell proliferation was detected by the Cell Counting-8 Kit (CCK-8) assay, and cell apoptosis was measured by flow cytometry. Targeted relationships among circ_DROSHA, miR-106b-5p and MEF2C were confirmed by a dual-luciferase reporter or RNA immunoprecipitation (RIP) assay. Our data showed that circ_DROSHA was down-regulated in the serum samples of TLE patients and the TLE cell model. Circ_DROSHA up-regulation alleviated the cytotoxicity of the TLE cell model by enhancing cell proliferation and repressing cell apoptosis. Circ_DROSHA directly bound to miR-106b-5p. Moreover, miR-106b-5p represented a downstream effector of circ_DROSHA function. MEF2C was a direct target of miR-106b-5p, and miR-106b-5p knockdown relieved magnesium-free treatment-induced cell injury by up-regulating MEF2C. Furthermore, circ_DROSHA regulated MEF2C expression via sponging miR-106b-5p. Our study suggested that the enforced expression of circ_DROSHA alleviated the cell damage of the TLE cell model at least in part through the regulation of the miR-106b-5p/MEF2C axis.     

lncRNA ZFPM2-AS1 promotes proliferation via miR-18b-5p/VMA21 axis in lung adenocarcinoma

Lung cancer has been proved to be one of the most common kinds of cancers around the globe. Meanwhile, as the predominant type of lung cancer, lung adenocarcinoma (LUAD) has received increasing attention in cancer research. Long noncoding RNAs (lncRNAs) are known to be associated with oncogenesis and progression of various cancers. However, many lncRNAs have not been thoroughly detected in LUAD. In this study, through bioinformatics analysis we found that zinc finger protein multitype 2 antisense RNA 1 (ZFPM2-AS1) was associated with poor prognosis of LUAD patients. Also, ZFPM2-AS1 was detected to be overexpressed in LUAD tissues and cells. Furthermore, ZFPM2-AS1 could promote the proliferation of LUAD cells. Next, miR-18b-5p was found to bind with and negatively regulated by ZFPM2-AS1. VMA21, target gene of miR-18b-5p, could bind with and be negatively regulated by miR-18b-5p. More importantly, both ZFPM2-AS1 and VMA21 were found to be attached to the RNA-induced silencing complex constructed from miR-18b-5p and Ago2. Also, ZFPM2-AS1 could regulate the expression of VMA21. Therefore, ZFPM2-AS1 were confirmed to regulate VMA21 by competitively binding with miR-18b-5p. Finally, rescue assays confirmed that ZFPM2-AS1 could regulate LUAD cell proliferation via miR-18b-5p/VMA21 axis.     

CircRNA circPDSS1 promotes the gastric cancer progression by sponging miR-186-5p and modulating NEK2

The aim of this study is to investigate the regulatory mechanism of circPDSS1/miR-186-5p/NEK2 axis on the viability and proliferation in gastric cancer (GC) cell line. Differentially expressed circRNAs, miRNAs, and mRNAs in GC tissues and paracarcinoma tissues were analyzed using gene chips GSE83521, GSE89143, and GSE93415. Then, the expression of circPDSS1, miR-186-5p, and NEK2 was analyzed via quantitative real-time polymerase chain reaction (qRT-PCR). Survival analysis was adopted to explore the association between the circPDSS1 expression and the prognosis of GC. The effect of circPDSS1 on GC cell cycle and apoptosis was verified with the flow cytometry. Targeting relationships among circPDSS1, miR-186-5p, and NEK2 were predicted via bioinformatics analysis and demonstrated by the dual-luciferase reporter assay. Our results showed that circPDSS1 and NEK2 were high-expressed whereas miR-186-5p was low-expressed in GC tissues and cells. CircPDSS1 promoted GC cell cycle and inhibited apoptosis by sponging miR-186-5p, while miR-186-5p inhibited cell cycle and promoted apoptosis by targeting NEK2. Thus, circPDSS1 acts as a tumor promoter by regulating miR-186-5p and NEK2, which could be a potential biomarker and therapeutic target for the management of GC.     

miR-216b-5p regulates proliferation and apoptosis of ox-LDL-stimulated VSMCs and HUVECs via IGF2

Atherosclerosis (AS) is one of the principal causes of cardiovascular disorder. Reportedly, vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) play key roles in AS development, and microRNAs (miRNAs) regulate their functions. The function of miR-216b-5p in AS remains unknown. Human VSMCs and human HUVECs were treated with ox-LDL to establish the in vitro model of AS. MiR-216b-5p and IGF2 expressions in VSMCs and HUVECs were probed by qRT-PCR and western blot. The viability, cell cycle progression, and apoptosis of VSMCs and HUVECs were evaluated by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine, and flow cytometry assays, respectively. The binding sites between IGF2 3′UTR and miR-216b-5p were validated by dual-luciferase reporter assay. miR-216b-5p expression was declined in ox-LDL-induced VSMCs and HUVECs. In VSMCs, miR-216b-5p overexpression inhibited excessive proliferation and induced apoptosis. MiR-216b-5p could markedly restrain the viabiblity of VSMCs induced by ox-LDL and enhanced the viability of HUVECs. Additionally, IGF2 was confirmed as the direct target of miR-216b-5p and transfection of IGF2 overexpression plasmids rescued the effects of miR-216b-5p on VSMCs and HUVECs. miR-216b-5p alleviates the dysfunction of VSMCs and HUVECs caused by ox-LDL via repressing IGF2, and exerts protective functions to block the development of AS.     

下调lncRNA MCF2L-AS1靶向miR-33b-5p抑制胃癌细胞增殖、迁移、侵袭并促进凋亡

摘要 目的：探讨lncRNA MCF2L-AS1对胃癌细胞恶性生物学行为的影响及分子机制。方法：选取45例胃癌患者的癌组织及癌旁正常组织,或培养胃黏膜上皮细胞GES-1、胃癌细胞HGC-27,采用RT-qPCR检测MCF2L-AS1和miR-33b-5p的表达水平。采用双荧光素酶报告实验检测MCF2L-AS1和miR-33b-5p的靶向关系。将HGC-27细胞分为si-NC组、si-MCF2L-AS1组、mimic NC组、miR-33b-5p mimic组、si-MCF2L-AS1+inhibitor NC组、si-MCF2L-AS1+miR-33b-5p inhibitor组,分别转染si-NC、si-MCF2L-AS1、mimic NC、miR-33b-5p mimic或共转染si-MCF2L-AS1+inhibitor NC、si-MCF2L-AS1+miR-33b-5p inhibitor。采用MTT实验检测细胞增殖情况,流式细胞术检测细胞凋亡率,克隆形成实验检测细胞克隆形成数,Transwell实验检测迁移和侵袭细胞数。结果：与癌旁正常组织或GES-1细胞相比,胃癌组织或HGC-27细胞中MCF2L-AS1表达水平升高、miR-33b-5p表达水平降低,差异均有统计学意义（P<0.05）。MCF2L-AS1可靶向调控miR-33b-5p。下调MCF2L-AS1或过表达miR-33b-5p,miR-33b-5p表达水平升高,HGC-27细胞凋亡率升高,但细胞增殖、克隆形成数、迁移和侵袭数均减少,差异均有统计学意义（P<0.05）。抑制miR-33b-5p可减弱下调MCF2L-AS1对HGC-27细胞的生物学作用。结论：下调MCF2L-AS1通过上调miR-33b-5p抑制胃癌细胞增殖、迁移、侵袭并促进凋亡;MCF2L-AS1通过靶向调控miR-33b-5p表达进而参与胃癌细胞的恶性生物学行为。     

Long noncoding RNA TCL6 binds to miR-106a-5p to regulate hepatocellular carcinoma cells through PI3K/AKT signaling pathway

Long noncoding RNAs (lncRNAs) have been reported to dysregulate and involve in the pathology of hepatocellular carcinoma (HCC). Nonetheless, the functional role of lncRNA T cell leukemia/lymphoma 6 (TCL6) and its underlying mechanism in HCC remain unclear. Herein, we analyzed the expression of TCL6 and elucidated its mechanistic involvement in HCC. Bioinformatics analyses indicated TCL6 was evidently downregulated in HCC tissues compared with normal controls. TCL6 was downregulated while microRNA-106a-5p (miR-106a-5p) was upregulated in HCC cell lines. Moreover, knockdown or overexpression of TCL6 significantly raised or diminished the expression level of miR-106a-5p in HCC cells, similar to the effect of miR-106a-5p on TCL6 expression. Functionally, TCL6 inhibited the proliferative, migratory, and invasive potentials of HCC cells as analyzed by cell counting kit-8, scratch wound healing, and transwell assays, respectively. Conversely, miR-106a-5p exerted an opposite effect on the proliferative, migratory, and invasive potentials of HCC. RNA immune precipitation and luciferase reporter assays revealed TCL6 directly bound to miR-106a-5p and luciferase reporter assay verified phosphatase and tensin homolog (PTEN) was a target gene of miR-106a-5p. Mechanistically, TCL6 knockdown evidently reduced PTEN expression at both messenger RNA and protein levels, and miR-106a-5p inhibitor partially rescued this reduction effect in HCC cells. Additionally, western blot assays demonstrated miR-106a-5p downregulation or TCL6 overexpression promoted the protein level of PTEN, and suppressed the phosphorylation level of AKT, the protein level of phosphatidylinositol 3-kinase (PI3K). Collectively, these results revealed TCL6 as a tumor-suppressive lncRNA regulates PI3K/AKT signaling pathway via directly binding to miR-106a-5p in HCC. This mechanism provides a theoretical basis for HCC pathogenesis and a potential therapeutic strategy for HCC treatment.     

microRNA-501-5p promotes cell proliferation and migration in gastric cancer by downregulating LPAR1

In spite of the achievement in treatment, the gastric cancer (GC) mortality still remains high. MicroRNAs (miRNAs) are a group of small noncoding RNAs that play a crucial part in tumor progression. In this study, we explored the expression and function of microRNA-501-5p (miR-501-5p) in GC cell lines. Quantitative real-time polymerase chain reaction assay results suggested that miR-501-5p was significantly upregulated in GC tissues and cell lines. And, the Cell Counting Kit-8 colony formation and cell migration assay results showed that the downregulation of miR-501-5p decreased GC cell proliferation and migration. Besides that, we found that GC cell cycle was arrested in G2 phase and cell apoptosis rate was increased by silencing the expression of miR-501-5p in GC cell lines using the flow cytometry. We also found that miR-501-5p could directly target lysophosphatidic acid receptor 1 (LPAR1) and negatively regulate LPAR1 expression in GC cell lines by performing dual-luciferase reporter gene assay and Western blot analysis. And, LPAR1 was significantly downregulated in GC tissues and inversely correlated with miR-501-5p expression. Furthermore, LPAR1 downregulation promoted cell proliferation and migration, which were attenuated by cotransfection of miR-501-5p inhibitor in GC cells. In conclusion, miR-501-5p can promote GC cell proliferation and migration by targeting and downregulating LPAR1. miR-501-5p/LPAR1 may become a potential therapeutic target for GC treatment.     

MiR-29a-5p inhibits proliferation and invasion and induces apoptosis in endometrial carcinoma via targeting TPX2

This study was aimed to explore the effects of miR-29a-5p expression and its target gene TPX2 (target protein for Xenopus kinesin-like protein 2) on endometrial cancer (EC) devel on EC development and to assess the prognostic impacts of TPX2. Microarray-based GEO and TCGA (the Cancer Genome Atlas) EC expression data were used to identify differentially expressed miRNAs and mRNAs. The observed potential target relationship between miR-29a-5p and TPX2 was verified using TargetScan and luciferase reporter assays. The mRNA and protein expression levels of miR-29a-5p and TPX2 were confirmed by qRT-PCR and western blot, respectively. Associations between TPX2 expression and patient prognosis were assessed using Kaplan-Meier and log-rank assays. Changes in EC-derived cell proliferation, invasion and apoptosis after exogenous miR-29a-5p and TPX2 over-expression and/or silencing were assessed using CCK-8 (cell counting kit-8), colony formation, Transwell and flow cytometry assays, respectively. We found that in primary EC tissues the expression of miR-29a-5p was down-regulated and the expression of TPX2 was up-regulated. We also found that low expression of TPX2 were associated with a better prognosis, and vice versa. Subsequent exogenous miR-29a-5p over-expression and TPX2 silencing could inhibit EC-derived cell proliferation and invasion, and to induce apoptosis. We also found that miR-29a-5p might target and repress TPX2, thereby inhibiting EC-derived cell proliferation and invasion and enhancing apoptosis. We conclude that miR-29a-5p could inhibit the proliferation and invasion of EC-derived cells and enhance the apoptosis of EC-derived cells via TPX2 down-regulation. A high TPX2 expression in primary EC tissues was found to be associated with a poor prognosis. As such, these biomarkers may serve as promising prognostic indicators.

ABBREVIATIONS: EC: Endometrial cancer; 3?-UTR: 3?-untranslated regions; TPX2: target protein for Xenopus kinesin-like protein 2; TCGA: the Cancer Genome Atlas; UCEC: uterine corpus endometrial carcinoma; CCK-8: cell counting kit-8; OD: optical density; FCM: flow cytometry; EMT: epithelial-mesenchymal transition     

Long noncoding RNA LINC00629 restrains the progression of gastric cancer by upregulating AQP4 through competitively binding to miR-196b-5p

Gastric cancer continues to be a common cancer in the world with high incidence and mortality. Accumulating evidence has implicated long noncoding RNAs (lncRNAs) in gastric cancer progression. Here, this study identified the potential role of a novel lncRNA, LINC00629 in gastric cancer and to elucidate the underlying mechanism. Initially, microarray-based gene expression profiling of gastric cancer was employed to identify differentially expressed genes. Next, the expression of LINC00629, microRNA-196b-5p (miR-196b-5p) and aquaporin 4 (AQP4) in clinical gastric cancer tissues was determined and the cell line presenting with the lowest LINC00629 expression was selected. The interaction among LINC00629, miR-196b-5p, and AQP4 was identified. Expression of LINC00629, miR-196b-5p, and AQP4 in gastric cancer cells were altered and then biological behaviors of gastric cancer cells were assessed by 5-ethynyl-2′-deoxyuridine and Transwell assays. Tumor formation in vivo was evaluated in nude mice. In gastric cancer, expression of LINC00629 and AQP4 was downregulated, and expression of miR-196b-5p was upregulated. Proliferation, invasion, and migration of gastric cancer cells were reduced after overexpression of LINC00629. LINC00629 competitively bound to miR-196b-5p, while AQP4 was a target of miR-196b-5p. Either downregulating miR-196b-5p or upregulating AQP4 could restrain the development of gastric cancer in vitro. LINC00629 overexpression repressed the growth of transplanted tumors in vivo. Taken together, LINC00629 competitively bound to miR-196b-5p to upregulate AQP4 expression, thereby inhibiting gastric cancer progression. Therefore, understanding of this mechanism may help to improve gastric cancer treatment.     

LncRNA PDCD4-AS1 alleviates triple negative breast cancer by increasing expression of IQGAP2 via miR-10b-5p

ObjectiveMounting evidence demonstrates that long non-coding RNA (lncRNA) is dysregulated in breast cancers. This study was designed to detect the influences and regulatory mechanism of lncRNA PDCD4-AS1 in triple-negative breast cancer (TNBC).MethodsqRT-PCR and Western blot were utilized to investigate the expression levels of PDCD4-AS1, miR-10b-5p and IQGAP2 in TNBC tissues and cells. Online software and luciferase reporter gene system were employed to testify the interactions among these molecules. Loss and gain of function of PDCD4-AS1, miR-10b-5p or IQGAP2 were performed before MTT and colony formation assay, TUNEL staining in addition to Transwell and scratch assays were applied to measure the cell biological functions.ResultsIn this work, PDCD4-AS1 and IQGAP2 were lowly expressed while miR-10b-5p was strongly expressed in TNBC tissues and cells. PDCD4-AS1 or IQGAP2 overexpression effectively attenuated TNBC cell proliferation, migration and invasion, and increased the apoptosis rate, while this effect was abandoned in response to miR-10b-5p mimics transfection. miR-10b-5p bound to IQGAP2 and acted as a downstream target of PDCD4-AS1.ConclusionOur findings identified lncRNA PDCD4-AS1 as a tumor suppressor in TNBC by regulating IQGAP2 expression via miR-10b-5p, giving a novel insight into the regulatory mechanism of PDCD4-AS1 in the pathogenesis of TNBC.     

鼻咽癌组织miR-20b-5p、miR-325-3p表达水平与放疗敏感性和预后的关系研究

摘要 目的：探讨鼻咽癌组织微小核糖核酸（miR）-20b-5p、miR-325-3p表达水平与放射治疗敏感性和预后的关系。方法：选取2017年11月至2019年6月我院收治的84例确诊为鼻咽癌并拟进行放射治疗的患者设为鼻咽癌组,另选取同期收治的42例慢性鼻咽炎患者为对照组,比较鼻咽癌组织及鼻咽部炎症组织中miR-20b-5p、miR-325-3p表达水平,分析鼻咽癌组织中miR-20b-5p、miR-325-3p表达水平与鼻咽癌患者临床病理特征的关系。根据鼻咽癌患者放疗敏感性评估结果分为敏感组和抵抗组,比较两组miR-20b-5p、miR-325-3p表达水平。随访3年,Kaplan-Meier法及Cox回归分析法分析miR-20b-5p、miR-325-3p表达水平与鼻咽癌患者生存预后的关系。结果：鼻咽癌组miR-20b-5p、miR-325-3p表达水平均高于对照组（P＜0.05）。不同T分期、N分期、临床分期患者在miR-20b-5p、miR-325-3p高表达组与低表达组中的占比比较存在统计学差异（P＜0.05）。完成7~8周放疗后3个月评估患者放疗抵抗率36.90%,抵抗组miR-20b-5p、miR-325-3p表达水平均高于敏感组（P＜0.05）。miR-20b-5p高表达鼻咽癌患者的累积生存时间短于miR-20b-5p低表达患者（P＜0.05）;miR-325-3p高表达鼻咽癌患者的累积生存时间短于miR-325-3p低表达患者（P＜0.05）。单因素、多因素Cox回归分析显示,年龄＞60岁、T3/T4期、miR-20b-5p高表达、miR-325-3p高表达是鼻咽癌患者预后不良的独立危险因素（P＜0.05）。结论：鼻咽癌组织中miR-20b-5p、miR-325-3p均异常高表达,其表达水平与肿瘤浸润深度、淋巴结转移、临床分期及放疗敏感性有关,且miR-20b-5p、miR-325-3p高表达患者放疗后预后不良风险更大。     

miR-135b-5p抑制脓毒症引起的小鼠急性肺损伤(ALI)的作用及机制*

目的: 探究miR-135b-5p在小鼠脓毒症(sepsis)引起的急性肺损伤(ALI)模型中的表达水平及其对小鼠肺部炎症反应和细胞焦亡的影响。方法: 将C57BL/6小鼠随机分为6组,每组8只,通过盲肠结扎穿刺法(CLP)手术构建CLP诱导的脓毒症小鼠模型:腹腔注射0.1 mg/kg的巴比妥麻醉,腹部纵向切开暴露盲肠,结扎盲肠并用注射器针头进行穿孔,挤出部分肠道内容物后缝合伤口。假手术组(Sham组)开腹后不做任何处理缝合伤口,无CLP手术处理。治疗组分为CLP+NC mimic组,CLP+miR-135b-5p mimic组,CLP+NC mimic+empty vector组,CLP+消皮素D (GSDMD)组,CLP+miR-135b-5p mimic+GSDMD组。治疗组小鼠在CLP手术前一周皮下注射200 μl溶解于生理盐水的NC mimic(200 nmol/L),miR-135b-5p mimic(200 nmol/L),empty vector(100 nmol/L),GSDMD vector(100 nmol/L),每天注射1次,连续一周。术后24 h采用二氧化碳窒息法实施安乐死。采用qRT-PCR检测小鼠肺组织样本中miR-135b-5p和GSDMD mRNA的表达水平;苏木精-伊红(HE)染色检测小鼠肺组织形态和损伤状态;采用5 ml生理盐水冲洗小鼠右肺3次,每次持续约3~5 min,收集肺泡灌洗液(BALF),酶联免疫吸附实验(ELISA)检测小鼠肺泡灌洗液(BALF)中GSDMD、白介素1β(IL-1β)和白介素18(IL-18)的表达水平;蛋白免疫印迹法检测小鼠肺组织内含NLR家族PYRIN域蛋白3(NLRP3),半胱氨酸天冬氨酸蛋白水解酶1(caspase 1)以及切割后的N-端GSDMD端蛋白结构域(cleaved-GSDMD-N)的表达水平。双荧光素酶报告基因检测系统验证miR-135b-5p与GSDMD的靶向结合关系。结果: 与对照组相比,CLP组小鼠肺组织中有大量的炎症细胞浸润,肺泡损伤,细胞间质水肿及肺泡塌陷等病理特征,小鼠肺组织内细胞焦亡相关蛋白(NLRP3,caspase-1和GSDMD)的表达水平明显增加(P<0.01),但miR-135b-5p的表达水平明显下调(P<0.01);与CLP组相比,超表达miR-135b-5p能够明显抑制CLP诱导的小鼠肺组织内细胞焦亡(P<0.01),靶向抑制GSDMD的表达水平(P<0.01);超表达GSDMD能够逆转超表达miR-135b-5p对肺组织细胞焦亡的抑制作用(P<0.01),超表达miR-135b-5p能够通过靶向GSDMD抑制小鼠BALF中IL-1β及IL-18的表达水平(P<0.01)。结论: miR-135b-5p靶向下调GSDMD抑制细胞焦亡,改善脓毒症引起的ALI,为脓毒症诱导的ALI治疗提供了潜在的治疗靶点和理论依据。     

长链非编码RNA IFNG-AS1靶向miR-19b-1-5p调控oxLDL诱导的血管内皮细胞增殖、凋亡的机制研究

为了探讨长链非编码RNA干扰素活化基因的反义核糖核酸(lncRNA IFNG-AS1)对氧化型低密度脂蛋白(oxLDL)诱导的人脐静脉血管内皮细胞EVC-304增殖、凋亡的影响和调控机制,该研究采用100μg/mL的oxLDL分别处理转染si-IFNG-AS1、miR-19b-1-5p mimics或共转染si-IFNG-AS1与anti-miR-19b-1-5p的EVC-304细胞,利用实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测细胞中IFNG-AS1和miR-19b-1-5p表达,细胞计数(CCK-8)法检测细胞增殖,流式细胞术检测细胞凋亡,蛋白免疫印迹(Western blot)检测细胞中细胞周期依赖性蛋白激酶抑制因子1A(P21)、多肿瘤抑制基因1(P16)、剪切的DNA修复酶(cleaved-PARP)、剪切的半胱天冬氨酸蛋白酶-3(cleaved-caspase-3)的蛋白表达情况;利用双荧光素酶报告基因实验验证IFNG-AS1和miR-19b-1-5p的靶向关系。结果显示,oxLDL促进了EVC-304细胞中IFNG-AS1的表达,而抑制了miR-19b-1-5p的表达(P<0.05);抑制IFNG-AS1或过表达miR-19b-1-5p提高了oxLDL处理的EVC-304细胞增殖活性及细胞中P21和P16蛋白表达,而降低了细胞凋亡率及cleaved-PARP和cleaved-caspase-3的蛋白表达(P<0.05);抑制miR-19b-1-5p逆转了抑制IFNG-AS1对oxLDL处理的EVC-304细胞增殖和凋亡的影响(P<0.05);双荧光素酶报告基因实验证实IFNG-AS1靶向调控miR-19b-1-5p表达。这提示抑制IFNG-AS1表达可促进oxLDL处理的EVC-304细胞增殖,并抑制细胞凋亡,其作用机制与靶向上调miR-19b-1-5p表达有关,IFNG-AS1/miR-19b-1-5p轴可能为动脉粥样硬化的治疗提供新的靶点。     

miR-193b-5p regulates chondrocytes metabolism by directly targeting histone deacetylase 7 in interleukin-1β-induced osteoarthritis

There is increasing evidence regarding the pivotal roles of microRNAs (miRNAs) and histone deacetylases (HDACs) in the development of osteoarthritis (OA). This study aimed to determine whether miR-193b-5p regulates HDAC7 expression directly to affect cartilage degeneration. Expression levels of miR-193b-5p, HDAC7, matrix metalloproteinase 3 (MMP3), and MMP13 were determined in normal and OA cartilage and primary human chondrocytes (PHCs) stimulated with interleukin-1β (IL-1β). PHCs were transfected with a miR-193b-5p mimic or inhibitor to verify whether miR-193b-5p influences the expression of HDAC7 and MMPs. A luciferase reporter assay was performed to demonstrate the binding between miR-193b-5p and the 3′-untranslated region (UTR) of HDAC7. Expression of miR-193b-5p was reduced in IL-1β-stimulated PHCs and in OA cartilage compared to that in normal cartilage. Luciferase reporter assay exhibited the repressed activity of the reporter construct containing the 3′UTR of HDAC7. Both miR-193b-5p overexpression and HDAC7 inhibition decreased the expression of MMP3 and MMP13, whereas the inhibition of miR-193b-5p enhanced HDAC7, MMP3, and MMP13 expression. miR-193b-5p downregulates HDAC7 directly and, as a result, inhibits MMP3 and MMP13 expression, which suggests that miR-193b-5p has a protective role in OA.     

miR-424-5p regulates apoptosis and cell proliferation via targeting Bcl2 in nucleus pulposus cells

ABSTRACT

miRNAs play an important role in the pathogenesis of intervertebral disc degeneration (IDD). The role and the underlying mechanism of miR-424-5p in human nucleus pulposus (NP) are still unknown. We aimed to explore the role of miR-424-5p in IDD.

Real-time PCR was used to detect the expression of miR-424-5p and Bcl2 in IDD tissues and idiopathic scoliosis tissues. Human NP cells were used in our study. MTT and Hoechst apoptosis assays were used to detect the proliferation and apoptosis of NP cells, respectively. Western blotting assays were used to detect the expression levels of Bcl-2, cleaved caspase-3, cleaved caspase-9, caspase-3 and caspase-9 in degenerative NP cells. A luciferase reporter assay was applied to confirm the relationship between miR-424-5p and Bcl2.

Our results showed that the expression of miR-424-5p was increased and Bcl2 was decreased in degenerative NP cells. miR-425-5p expression was negatively correlated with Bcl2 expression in IDD tissues. Suppression of miR-424-5p using an inhibitor increased Bcl2 expression at both the mRNA and protein levels, and it promoted cell viability and inhibited apoptosis. Furthermore, the levels of cleaved caspase-3 and cleaved caspase-9 were downregulated in miR-424-5p-silenced NP cells. Interestingly, we found that silencing miR-424-5p increased p62 expression at both the mRNA and protein levels. Finally, a luciferase reporter assay verified the binding of the miR-424-5p and the 3’UTR of Bcl2.

These results suggested that silencing miR-424-5p suppressed NP cell apoptosis by upregulating Bcl2. Therefore, miR-424-5p might be a novel target for IDD therapies.     

Cucurbitacin B suppresses proliferation of pancreatic cancer cells by ceRNA: Effect of miR-146b-5p and lncRNA-AFAP1-AS1

Cucurbitacin B (CuB) is a natural tetracyclic triterpene product that displays antitumor activity against a wide variety of cancers. In this study, we explored the antipancreatic cancer activity of CuB via the inhibition of expression of the cancer-related long noncoding RNA, actin filament-associated protein 1-antisense RNA 1 (AFAP1-AS1). CuB arrested pancreatic cancer (PC) cells in the G2/M cell cycle phase by suppressing the expression of AFAP1-AS1. Insights into the mechanisms of competing endogenous RNAs (ceRNAs) gained from bioinformatics analysis and luciferase activity assays showed that the epidermal growth factor receptor (EGFR) and AFAP1-AS1 directly compete for miR-146b-5p binding. CuB-induced high miR-146b-5p expression and inhibited the expression of AFAP1-AS1. In summary, reducing the expression of endogenous AFAP1-AS1 effectively increased the available concentration of miR-146b-5p in PC, whereas miR-146b-5p overexpression prevented the expression of endogenous AFAP1-AS1. In particular, we hypothesized that AFAP1-AS1 might act as a ceRNA, effectively becoming a sponge for miR-146b-5p, thereby activating the expression of the EGFR. Thus, CuB suppresses the proliferation, in vitro and in vivo, of PC cells through the ceRNA effect of AFAP1-AS1 on miR-146b-5p.     

miR-216b-5p靶向调控BTN3A2促进胶质瘤细胞LN-229的迁移以及侵袭能力

大量证据表明microRNA（miRNA）通过靶向调控靶基因的表达从而在肿瘤侵袭与转移中发挥重要作用。然而关于microRNA-216b-5p (miR-216b-5p )通过靶向嗜乳脂蛋白第3亚家族膜蛋白A2（butyrophilin subfamily 3 member A2,BTN3A2）促进胶质瘤侵袭与转移的机制尚不明确。本研究通过GSE15824与GSE4290差异表达分析筛选出同时在2个芯片中表达上调的BTN3A2（P＜0.05）。生存曲线结果显示,高表达BTN3A2病人总生存期明显下降（P＜0.001）。表达量分析结果显示,BTN3A2表达随WHO分级升高而升高（P＜0.05）,同时1p/19q未联合缺失与IDH突变型病人BTN3A2表达升高（P＜0.001）。基因集富集分析（gene set enrichment analysis,GSEA）结果显示,BTN3A2与众多癌症相关通路有关（P＜0.05）;Western印迹结果显示,BTN3A2在7例胶质瘤组织和胶质瘤细胞系U87、U251和LN-229中表达上调,过表达miR-216b-5p （miR-216b-5p mimics）后BTN3A2蛋白表达水平降低;Transwell结果显示,转染BTN3A2干扰质粒（si-BTN3A2）和miR-216b-5p mimics后可以抑制LN 229细胞体外迁移与侵袭能力（P＜0.05）;在线预测网站证实,miR-216b-5p 为BTN3A2潜在靶基因;生存曲线结果显示,与低表达miR-216b-5p 病人相比,高表达病人生存率明显上调（P=0.025）;荧光定量RT PCR结果显示,miR-216b-5p 在胶质瘤U87、U251和LN-229细胞中表达下降（P＜0.05）;双荧光素酶结果显示,BTN3A2存在与miR-216b-5p 的结合靶点(P<005);综上所述,BTN3A2可能通过结合miR-216b-5p 促进胶质瘤细胞LN 229的迁移以及侵袭。     

miR-106b-93-25簇对子宫内膜癌细胞的调控作用研究

目的:检测mi R-106b-93-25基因簇对子宫内膜癌细胞增殖及凋亡的影响,并探讨其机制。方法:q RT-PCR检测临床子宫内膜癌标本及癌旁正常组织中mi R-106b、mi R-93和mi R-25及其宿主基因MCM7的表达情况。将micro RNA及其拮抗剂转染ECC-1细胞后,MTT实验检测ECC-1细胞增殖情况,流式细胞术检测ECC-1细胞周期及细胞凋亡情况。荧光素酶报告系统验证mi R-106b和mi R-25分别直接调控p21和Bim。结果:临床标本子宫内膜癌组织与癌旁正常组织相比mi R-106b-93-25簇及其宿主基因MCM7的表达明显增高。mi R-106b-93-25簇能够促进ECC-1细胞增殖,减少凋亡。转染mi R-106b和mi R-93的细胞出现明显的S期阻滞,过表达mi R-25的细胞凋亡明显减少。mi R-106b-93-25簇通过抑制靶基因p21和Bim的表达,引起促增殖、抗凋亡作用。结论:mi R-106b-93-25簇能够促进子宫内膜癌细胞增殖,抑制凋亡,并使细胞发生S期阻滞。mi R-106b-93-25簇在子宫内膜癌的发生与发展中具有重要的作用。